Our preliminary data show that opioid drugs, administered to sickle patients promote growth of microvascular endothelial cells. This opioid effect on endothelium is accompanied by MAPK/ERK activation and promotion of in vitro (tube formation) and in vivo (in Matrigel implants in mice) angiogenesis. We hypothesize that administration of opioids to sickle patients may increase their risk for retinopathy by pro-angiogenic signaling of opioids in endothelium. We will test this via 4 Specific Aims. I Aim#1 Characterize opioid induced endothelial proliferation, by testing specific hypotheses that endothelial growth response to opioids is, [a] exaggerated for human dermal microvascular endothelial cells (HDMEC) and retinal EC vs. HUVEC, [b] influenced by pro-inflammatory cytokines and VEOF, [c] accompanied by EC activation, cell adhesion molecule and NOS expression, Ed] caused by both a stimulation of growth and inhibition of apoptosis. Aim#2 Identify mechanism of endothelial growth stimulation by opioids via 4 specific hypotheses, that [a] opioid stimulated growth involves specific opioid receptors in both presence and absence of pro-inflammatory cytokines, [b] pro-inflammatory cytokines and growth factors regulate mu opioid receptor (MOR) expression on human microvascular EC, [c] opioid signaling in endothelium involves MAPK/ERK phosphorylation via Gi coupled receptors and NO, Ed] opioid induced proliferation is I dependent upon above signaling pathway. Aim#3 Determine if pro-inflammatory cytokine induced MOR. expression and opioid-induced endothelial growth actually promotes angiogenesis in vitro (tube formation in Matrigel) and in vivo (in Matrigel implants in mice). Aim #4 Use sickle mice to determine if opioids exert biologically important effects in vivo, by testing specific hypotheses that administration of opioids to sickle mice [a) causes increased endothelial activation and cell adhesion molecule and NOS expression, [b] accelerates and/or exaggerates development of retinopathy, [c] improves rate of healing of skin wounds; (longer-term goal). Mostly HDMEC and some retinal microvascular EC will be used, since it is critical for angiogenesis studies to use microvascular not large vessel EC. If our hypotheses are true, this Project may lead to several implications of opioid signaling, resulting in altered, clinical decision making and perhaps even to development of novel therapeutics.